Eukaryotes employ phosphorylation and dephosphorylation of specific proteins to regulate many cellular processes (T. Hunter, Cell80:225-236 (1995); (Karin, M., Curr. Opin. Cell Biol. 3: 467-473 (1991)). These processes include signal transduction, cell division, and initiation of gene transcription. Thus, significant events in an organism's maintenance, adaptation, and susceptibility to disease are controlled by protein phosphorylation and dephosphorylation. These phenomena are so extensive that it has been estimated that humans have around 2,000 protein kinase genes and 1,000 protein phosphatase genes (T. Hunter, Cell80:225-236 (1995)), some of these likely coding for disease susceptibility. For these reasons, protein kinases and phosphatases are good targets for the development of drug therapies.
The most frequently used protein kinase and phosphatase screens employ either radioactive ATP or ELISAs. However, the use of radioactive ATP is undesirable due to the attendant costs of record-keeping, waste-disposal, and the fact that the assay format is not homogeneous. ELISAs are undesirable because they have a lower assay throughput due to the extra steps required for both washing and the enzyme reaction.
Fluorescence detection in the visible wavelengths offer an alternative to the use of radiotracers or ELISAs for kinase and phosphatase assays, as fluorescence offers detection limits comparable to those of radioactivity. Furthermore, this eliminates the cost of radioactive waste disposal. For example, the change in absorbance and fluorescence spectra of phosphotyrosine which occurs upon dephosphorylation has been used for the continuous monitoring of protein-tyrosine phosphatase (PTP) activity (Zhao, Z. et al., Anal. Biochem. 202:361-366 (1993)). However, previously developed fluorometric assays for kinases and phosphatases have not been especially amenable to the requirements of high throughput screening.
Fluorescence detection frequently offers the advantage of using homogeneous assay formats (i.e.--"mix, incubate, and read"). Indeed, the high throughput screening (HTS) field is moving rapidly toward the use of fluorescence, luminescence, absorbance, and other optical methods. Two fluorescence techniques, fluorescence polarization (FP) and fluorescence resonance energy transfer (FRET) are finding widespread use for assays, both in the private sector for HTS, secondary assays including kinetics, SAR studies, etc., and in university laboratories. The use of FP is particularly desirable since its readout is independent of the emission intensity (Checovich, W. J., et al., Nature 375:254-256 (1995); Dandliker, W. B., et al., Methods in Enzymology 74:3-28 (1981)) and is thus insensitive to the presence of colored compounds that quench fluorescence emission. FRET, although susceptible to quenching, can also be used effectively, especially for continuous enzyme assays.
From the forgoing, it will be clear that there is a continuing need for the development of cost-effective, facile, and sensitive optical kinase and phosphatase assays for both high throughput screening (HTS) and secondary assays.